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WO1995016697A1 - Conglutinine bovine recombinee et fragments de celle-ci - Google Patents

Conglutinine bovine recombinee et fragments de celle-ci Download PDF

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Publication number
WO1995016697A1
WO1995016697A1 PCT/US1994/014656 US9414656W WO9516697A1 WO 1995016697 A1 WO1995016697 A1 WO 1995016697A1 US 9414656 W US9414656 W US 9414656W WO 9516697 A1 WO9516697 A1 WO 9516697A1
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WO
WIPO (PCT)
Prior art keywords
peptide
protein
conglutinin
amino acid
acid sequence
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US1994/014656
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English (en)
Inventor
Young Moo Lee
Kevin R. Leiby
Thomas B. Okarma
Kedarnath Sastry
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Boston University
Applied Immune Sciences Inc
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Boston University
Applied Immune Sciences Inc
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Publication date
Application filed by Boston University, Applied Immune Sciences Inc filed Critical Boston University
Priority to AU14404/95A priority Critical patent/AU1440495A/en
Publication of WO1995016697A1 publication Critical patent/WO1995016697A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals

Definitions

  • the invention is directed to defined protein and peptide molecules and recombinant materials for their preparation. More particularly, the invention concerns recombinant bovine conglutinin and fragments thereof.
  • conglutininin The isolation of conglutinin from bovine plasma was described in 1967 by Lachmann, P.J. Adv Immunol (1967) 6 . :480-527. The activity of this protein was originally described in 1906 as causing agglutination of erythrocytes that have reacted with antibody and complement. Subsequent structural studies have elucidated the structure of this protein. Davis, III, A.E. et al . Biochemistry (1984) 22:2139-2144 describe studies which characterize the isolated single polypeptide chain of conglutinin as having a molecular weight of 48 kD.
  • the conglutinin consists of two domains: a collagenous N-terminal domain and a lectin- type C-terminal domain.
  • This single peptide is associated in vivo as a complex of six disulfide-linked polypeptide chains to provide a complex of molecular weight of about 300 kD.
  • the ultrastructure of the protein was reported on the basis of electron microscopic studies by Strang, A.J. et al. Biochem J (1986) 234:381-389. The homology of various portions of bovine conglutinin with other proteins has been explored by Young, N.M. et al. Biochem Biophys Res Comm (1987) 143 :645-651. Conglutinin is also found in human serum (Baatrup, G. et al . Scand J Immunol (1987) 26 :355-361; Thiel, S.
  • the protein consists of 351 amino acid residues and contains a 171-residue collagenous domain which separates a short noncollagenous N-terminal region of 25 residues from a 155-residue globular carboxy- terminus which shows a structural relationship with mannose-binding proteins and is responsible for the lectin activity.
  • conglutinin has been implicated in the inhibition of Human Immunodeficiency Virus-I by Ushigima, H. et al . Japan J Cancer Res (1992) £2:458-464 and the bovine form of the protein was shown to inhibit interaction of HIV-I envelope glycoprotein gpl60 with CD4 by Andersen, 0. et al . Scand J Immunol (1991) 33 :81-88.
  • Conglutinin has been shown to bind specifically to monocytes and macrophage as well as other cell types through its ability to couple to the Clq receptor present on these cells. This capacity has been noted in articles by Malhotra, R. et al.
  • Immobilized bovine conglutinin has also been used for the extracorporeal removal of immune complexes as described by Pitt, A.M. et al . International J Artificial Organs (1980) 2:42-49.
  • bovine conglutinin and defined fragments thereof to behave as lectins in specifically binding carbohydrate moieties makes this protein and its fragments useful in the design of materials which can be used to selectively deplete samples of, or to purify from biological samples containing them, materials which are characterized by the carbohydrates to which the bovine conglutinin lectin functionality binds.
  • the availability of recombinant materials for production of this characteristic lectin region provides the opportunity to design techniques for these procedures. Disclosure of the Invention
  • the invention provides recombinant materials which are useful in various separation procedures where the materials to be separated contain carbohydrate functionalities or moieties.
  • the invention materials are related to bovine conglutinin and can be produced recombinantly.
  • the invention is directed to recombinant materials for the production of conglutinin, especially bovine conglutinin, including purified and isolated DNA molecules containing nucleotide sequences encoding relevant portions of the conglutinin amino acid sequence, expression systems, cells modified to contain the expression systems and methods for recombinant production.
  • the invention is also directed to solid supports to which conglutinin- related fragments have been bound covalently, which are useful in the separation of materials having carbohydrate moieties bound by the lectin portion of conglutinin.
  • the collagenous region is also useful to bind cells displaying Clq receptor.
  • Figure 1 shows the nucleotide sequence and deduced amino acid sequence of a cDNA encoding bovine conglutinin.
  • the invention provides recombinant materials for the production of carbohydrate binding portions of the bovine conglutinin protein. Some of these ' recombinant materials are also useful in regulating the expression of genes encoding conglutinin either in recombinant host cells, in in vi tro systems, or in intact organisms expressing these genes.
  • the amino acid sequence of the lectin-like portion of bovine conglutinin has been elucidated, the production of the relevant fragments or proteins can be achieved, not only using recombinant techniques, but also by using synthetic methods for the production of peptides including both solution phase and solid phase synthesis. Both of these procedures are well understood in the art, and solid phase synthesis can be conducted using commercially available equipment and reagents.
  • Figure 1 shows the nucleotide sequence and deduced amino acid sequence of bovine conglutinin.
  • the protein is produced including a presequence of 20 amino acids which is cleaved to obtain the mature protein representing amino acids 1- 351 in Figure 1.
  • the residues at positions 26-196 represent a collagen-type domain with the repeating sequence GXY. This portion of the molecule also contains several hydroxylysine residues which are conveniently used for coupling to solid supports.
  • the C-terminal domain beginning at approximately residue 197 appears to be responsible for the lectin-like characteristics of the molecule.
  • the region at positions 250-351 shares considerable homology with other animal lectins. Accordingly, this region of the molecule, and carbohydrate binding regions thereof, are useful in separation of carbohydrate-bearing moieties.
  • residues shown at positions 250-351, or fragments thereof containing preferably 10 or more, more preferably 15 or more and most preferably 25 or more amino acid residues which demonstrate the capability to bind relevant carbohydrates.
  • Such ability can readily be ascertained by coupling the fragment to a solid support, labeling the moiety whose binding is to be tested, and assessing the ability of the support to retain the label.
  • Such simple techniques for ascertaining affinity are well understood in the art .
  • control sequences for prokaryotes have been known for more than ten years .
  • These expression systems can use constitutive or inducible promoters, including hybrid promoters such as the commercially available TRC promoter. Suitable
  • oligonucleotides useful in recombinant production, therapeutic and diagnostic contexts. Modulation of the expression of the gene encoding bovine conglutinin is useful in regulating the production of the recombinant material as well as modulating immune responses in si tu . In addition, assessing the level of RNA present in the liver which corresponds to the conglutinin molecule is an index to the response of the subject to infection.
  • oligomers based on the nucleotide sequence disclosed in Figure 1 herein can be used in standard assay methods for detecting the conglutinin-encoding DNA or RNA.
  • oligomers "based on” the sequence disclosed in Figure 1 is meant oligomers that contain portions of this sequence, that are complementary to the sequence or portions thereof, that represent primers used to amplify portions of the sequence when large amounts of DNA are desirable (such as for genetic manipulation) as well as oligomers designed on the basis of the disclosed sequence which, for example, effect triple helix formation with the relevant portion . of the duplex representing the conglutinin gene.
  • Relevant design parameters for PCR primers, oligomers capable of hybridizing to single strand targets, and oligomers capable of triple helix formation with DNA duplexes are well known in the art.
  • oligomers "based on” the DNA of Figure 1 may have the same sequence as a portion of this DNA, the same sequence as the complement or portion thereof, or a different sequence but one which corresponds to that disclosed in Figure 1 through art-known design parameters.
  • the oligomers having nucleotide sequences based on the nucleotide sequence shown in Figure 1 may be conventional RNA or DNA polymers, or may be modified forms thereof as generally known in the art .
  • the phosphodiester bonds of the oligomers may be substituted by alternative linkages such as phosphorothioates, methylphosphonates and the like.
  • alternative scaffolding for nucleotide bases has also been disclosed and such modifications are included within the scope of oligomers claimed herein.
  • the recombinantly reproduced peptides and proteins of the invention find utility in performing separations where the substrates to be separated are marked by the relevant carbohydrates.
  • immune complexes contain appropriate sugars so that they can be bound to the lectin portion of the conglutinin molecule. While the complete bovine conglutinin has been used in this manner, as described by Pitt, A.M. et al . International J Artificial Organs (1980) 2:42-49, as set forth above, the availability of the amino acid sequence of the relevant portion of the molecule, as well as the recombinant materials related to it permit design of more efficient systems for conducting such separations.
  • the protein or peptide containing a sequence of amino acids which confers ability to bind the relevant carbohydrates will be bound to a solid support, such as beads, microtiter trays, or other solids used generally in chromatography for use in the invention methods.
  • a solid support such as beads, microtiter trays, or other solids used generally in chromatography for use in the invention methods.
  • Such coupling can be achieved through standard derivatization of the support, depending on its chemical nature, and coupling of the derivatized support to, for example, sulfhydryl groups, amino groups, or carboxyl groups of the relevant protein.
  • the peptide or protein is coupled to solid support in an appropriate density for the application desired and optimization of such parameters is well known in the art.
  • the sample is contacted with the derivatized support for a time sufficient, and under conditions which are appropriate to, the binding of the immune complex to the peptide or protein ligand attached to the support.
  • the support is removed, then, from the sample and the remaining unadsorbed sample is depleted in the concentration of the immune complex.
  • the treated support to which the immune complex is adsorbed is eluted with a suitable eluant .
  • Suitable eluants include, in particular, competitive carbohydrate moieties which displace the immune complex from the solid support, and chelating agents such as EDTA which remove Ca +2 , essential for lectin function.
  • the peptide or protein ligands themselves are susceptible to considerable design parameters. It is believed that the ligand of the bovine conglutinin shown in Figure 1 representing positions 250-351 inclusive is intimately involved in binding of the carbohydrates. Thus, this section of the molecule is particularly relevant to construction of the invention ligands. Smaller portions of this region may also be used as long as their binding characteristics are retained. It may be advantageous to provide peptides or proteins which have more than one carbohydrate binding sequence; for example, a peptide could be constructed which has three contiguous copies of the amino acid sequence represented by positions 250-300 or a peptide which has two such regions and an additional region representing positions 275-325. As set forth above, the capability of the constructed ligand to bind the carbohydrate marked moiety can readily be tested by using either labelled carbohydrate moiety or a competition assay for binding of such moiety.
  • conglutinin levels produced in individuals may also be useful to assess conglutinin levels produced in individuals as an index to a response to infection.
  • This can be performed on biological samples, for example, using antibodies raised with respect to bovine conglutinin.
  • antibodies need not be formed in response to the complete conglutinin molecule, but rather can be formed from suitable epitopes derived from the amino sequence. It may be necessary to couple smaller epitopes to carrier in order to enhance immunogenicity.
  • antisera raised using standard immunization protocols, or monoclonal antibodies prepared from peripheral B cells or splenocytes of immunized animals can be used to conduct immunoassays for conglutinin levels in biological samples using standard immunoassay procedures .
  • the level of conglutinin production can also be assessed by conducting suitable assays for messenger RNA in the liver. As described below, liver appears to be the only tissue in which conglutinin is made.
  • the oligomers of the invention based on the oligonucleotide sequence shown in Figure 1 may be useful as probes in Northern blot assays, although as indicated below, it is preferred to use RNase based assays to detect the presence of mRNA. Oligomers, in particular, antisense oligonucleotide sequences based on the nucleotide sequence of Figure 1 are also useful in this assay, as described in Example 1.
  • oligomers designed to modulate the production of conglutinin may be useful in modulating the response of subjects to infection as well as in regulating the production of conglutinin related peptides and proteins using recombinant methods .
  • the following examples are intended to illustrate but not to limit the invention.
  • the PCR parameters consisted of one initial cycle of denaturation at 95° for 5 min, annealing at 55° for 5 min, and extension at 72° for 3 min followed by 40 cycles of denaturation at 95° for 1 min, annealing at 55° for 1 min, and extension at 72° for 3 min. The final cycle was a 7 min extension at 72°.
  • the reaction mixture (50 ⁇ l) contained 10 pmol of each primer, 0.2 mM dNTP, 2.5 U Taq polymerase, IX PCR reaction buffer (Perkin Elmer Cetus, Norwalk, CT) , and lug of bovine genomic DNA. A PCR product of 186 bp was obtained.
  • RNA from various bovine tissues was hybridized to a riboprobe obtained by transcription of a 260 bp PstI genomic fragment encoding a portion of the CRD exon inserted in a KS + host vector prepared as follows : the PCR product was labeled using random hexamers with 32 P dCTP, and used to screen a bovine genomic library in cosmid vector pWE15 (Stratagene, La Jolla, CA) . One clone, 16-2 was used for further study. A 260 bp PstI fragment from this genomic clone was subcloned into KS + vector (Stratagene, La Jolla, CA) .
  • the 32 P riboprobe (Specific activity 7.58 x 10 8 ) derived by transcription using T3 polymerase with maxiscript kit (Ambion, Austin, TX) , was used for a RNase protection assay following instructions provided (RPA II kit, Ambion, Austin, TX) .
  • RNase protection assay following instructions provided (RPA II kit, Ambion, Austin, TX) .
  • Approximately 1.7 x 10 5 cpm probe was hybridized with either total RNA from bovine brain, heart, jejunum, lung, rumen and spleen (20 ⁇ g each) or poly A+ RNA from bovine kidney and liver (1 ⁇ g each) .
  • the jejunum and rumen total RNA were isolated by the modified single step procedure (Chomczynski, P. et al .
  • RNAzol B Biotex, Houston, TX
  • Yeast tRNA (20 ⁇ g) served as control.
  • the full length PstI riboprobe along with polylinker sequences was 334 bp long and the expected size of protected fragments was 260 bp.
  • the RNase digests were run on 5% acrylamide-urea gel and were exposed to X-ray film with intensifying screens at -70°C for 96 hours. The results indicated that bovine liver is the only tissue expressing bovine conglutinin mRNA.
  • Example 2 Isolation and Characterization of Bovine Conglutinin cDNA The 186 bp PCR fragment, described in Example
  • the master plates are incubated at room temperature overnight to allow additional growth of the bacterial colonies and are then stored in the refrigerator.
  • the replica membranes with bacterial colonies are lysed and processed, following the protocol suggested by the manufacturer of the membranes (Hybond N + , Amersham, Arlington Heights, IL) .
  • the objective of this process is to immobilize the cosmid and bacterial DNA onto nylon membranes.
  • 5X SSPE is 0.18M NaCl, lOmM sodium phosphate, pH7.7 (Na 2 HP0 4 is added to NaH
  • the heat denatured 32 P labeled 186bp PCR fragment was then added to prehybridization solution and the incubation is continued overnight in a shaking water bath at 65°C.
  • the membranes were next washed for 30 min in 2X SSC (IX SSC is 0.15M NaCl/0.015M sodium citrate) /0.1% (w/v) SDS at room temperature, followed by 30 min in IX SSC/0.1% (w/v) SDS at 65°C.
  • the nylon membranes were exposed to X-ray film overnight (approx. 16 hours) at -70°C, with intensifying screens to detect colonies harboring the bovine conglutinin gene cosmid.
  • 16-2 One of the genomic cosmid clones isolated, called 16-2, was studied further and a 550bp X oI-Ba_ ⁇ _HI genomic fragment (encoding the amino terminal end of the bovine conglutinin) was subcloned from 16-2 cosmid into KS + vector and was used as a probe to screen lambda phages from a 5' stretch bovine liver cDNA library (Clontech, Palo Alto, CA) . The lambda phage library of bovine liver cDNA was screened following the procedures recommended by the manufacturer (Clontech) .
  • the method for screening the phage library is essentially similar to the procedure described above for the isolation of genomic clone, except that the cDNA is inserted into a phage, which multiplies inside bacterial cells, eventually killing them, causing appearance of 'plaques' .
  • the cDNA is inserted into a phage, which multiplies inside bacterial cells, eventually killing them, causing appearance of 'plaques' .
  • the clone clones screened only one clone was found to hybridize to the probe. Restriction analysis of the clone insert revealed three EcoRI fragments of approximately 1.6, 0.6 and 0.5 kb, respectively. The two small EcoRI fragments were cloning artifacts since they had 100% identity to mouse and human 28 S rRNA.
  • the 1.6 kb EcoRI fragment that encodes bovine conglutinin was sequenced in its entirety on both the strands, by subcloning various fragments into plasmid vectors.
  • the isolated bovine conglutinin cDNA clone has a 230 bp 5' untranslated region, a 1116 bp coding region which is then followed by a 173 bp 3' untranslated region.
  • the complete nt sequence of the isolated bovine conglutinin cDNA clone is shown in Figure 1.
  • the third and the longest ORF from the 5' end of the cDNA clone encodes a signal peptide and an amino acid sequence which is identical to the previously determined bovine conglutinin amino acid sequence, except two differences in the collagen domain: His 153 , Lys 190 in place of Arg and Ser respectively (Lee, et al . (1991) supra) .

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Abstract

Des matériaux recombinés apparentés à la conglutinine bovine fournissent des moyens de préparer des réactifs intéressants pour l'isolement ou l'épuisement de fractions contenant des résidus d'hydrates de carbone. Plus particulièrement, ces matériaux sont utiles pour isoler les complexes immuns ou épuiser des échantillons de ceux-ci.
PCT/US1994/014656 1993-12-16 1994-12-14 Conglutinine bovine recombinee et fragments de celle-ci Ceased WO1995016697A1 (fr)

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AU14404/95A AU1440495A (en) 1993-12-16 1994-12-14 Recombinant bovine conglutinin and fragments thereof

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US16845893A 1993-12-16 1993-12-16
US08/168,458 1993-12-16

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997007210A1 (fr) * 1995-08-17 1997-02-27 Fuso Pharmaceutical Industries, Ltd. Conglutinine recombinee et son procede de production
US6110708A (en) * 1995-08-17 2000-08-29 Fuso Pharmaceutical Industries, Ltd. Recombinant conglutinin and producing method thereof

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4906734A (en) * 1985-12-10 1990-03-06 Novo Industri A/S Human conglutinin
US5190931A (en) * 1983-10-20 1993-03-02 The Research Foundation Of State University Of New York Regulation of gene expression by employing translational inhibition of MRNA utilizing interfering complementary MRNA

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5190931A (en) * 1983-10-20 1993-03-02 The Research Foundation Of State University Of New York Regulation of gene expression by employing translational inhibition of MRNA utilizing interfering complementary MRNA
US4906734A (en) * 1985-12-10 1990-03-06 Novo Industri A/S Human conglutinin

Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
ARCH. BIOCHEM. BIOPHYS., Volume 305, No. 2, issued September 1993, N. KAWASAKI et al., "Differentiation of Conglutination Activity and Sugar-Binding Activity of Conglutinin After Removal of NH2-Terminal 54 Amino Acid Residues by Endogenous Serine Proteases", pages 533-540. *
BIOCHEM. BIOPHYS. RES. COMM., Volume 191, No. 2, issued 15 March 1993, Y. SUZUKI et al., "Cloning and Sequencing of a cDNA Coding for Bovine Conglutinin", pages 335-342. *
BIOCHEM. J., Volume 292, Pt. 1, issued 1993, J. LU et al., "The cDNA Cloning of Conglutinin and Identification of Liver as a Primary Site of Synthesis of Conglutinin in Members of the Bovidae", pages 157-162. *
CHEMICAL REVIEWS, Volume 90, No. 4, issued June 1990, E. UHLMANN et al., "Antisense Oligonucleotides: a New Therapeutic Principle", pages 544, 545, 575. *
IMMUNOLOGY, Volume 76, No. 1, issued May 1992, U. HOLMSKOV et al., "Tissue Localization of Conglutinin, a Bovine C-Type Lectin", pages 169-173. *
IMMUNOLOGY, Volume 78, No. 1, issued January 1993, B.L. LIM et al., "Structural Similarity Between Bovine Conglutinin and Bovine Lung Surfactant Protein D and Demonstration of Liver as a Site of Synthesis of Conglutinin", pages 159-165. *
J. BIOL. CHEM., Volume 266, No. 5, issued 15 February 1991, Y.M. LEE et al., "Primary Structure of Bovine Conglutinin, a Member of the C-Type Animal Lectin Family", pages 2715-2723. *
SCAND. J. IMMUNOL., Volume 36, No. 1, issued July 1992, O. ANDERSEN et al., "Purification, Subunit Characterization and Ultrastructure of Three Soluble Bovine Lectins: Conglutinin, Mannose-Binding Protein and the Pentraxin Serum Amyloid P-Component", pages 131-141. *

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997007210A1 (fr) * 1995-08-17 1997-02-27 Fuso Pharmaceutical Industries, Ltd. Conglutinine recombinee et son procede de production
AU717207B2 (en) * 1995-08-17 2000-03-23 Fuso Pharmaceutical Industries, Ltd. Recombinant conglutinin and producing method thereof
US6110708A (en) * 1995-08-17 2000-08-29 Fuso Pharmaceutical Industries, Ltd. Recombinant conglutinin and producing method thereof
EP0846701A4 (fr) * 1995-08-17 2001-02-07 Fuso Pharmaceutical Ind Conglutinine de recombinaison et procede pour produire cette substance
US6365342B1 (en) 1995-08-17 2002-04-02 Fuso Pharmaceutical Industries, Ltd. Methods for detecting anti-viral activity of calcium-dependent lectins
KR100385538B1 (ko) * 1995-08-17 2003-10-04 후소 야쿠힝 고교 가부시끼가이샤 칼슘-의존성렉틴을이용한항바이러스활성의검출방법
KR100395065B1 (ko) * 1995-08-17 2003-11-28 후소 야쿠힝 고교 가부시끼가이샤 재조합콘글루티닌및그의제조방법
US6979727B2 (en) 1995-08-17 2005-12-27 Fuso Pharmaceutical Industries, Ltd. Recombinant conglutinin and producing method thereof

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